Post on 25-May-2015
description
PRESENTED BY- SYED FAYYAZUDDIN PHARMACOLOGY
POLYMERASE CHAIN REACTIONPOLYMERASE CHAIN REACTION is a
laboratory technique for generating large number of a specified DNA.
“copying machine”
“cell free amplification technique”
HISTORYIn the year 1971 in the journal of molecular
biology Kleppe and co-workers has described a method using an enzymatic assay to replicate a short DNA template with primers.
However, this early manifestation of the basic PCR principle did not receive much attention.
This technique was invented by Kary Mullis.
PRINCIPLEThe purpose of PCR is to make huge number
of copies of gene.There are three major steps in a PCR, which
are repeated for 30 or 40 cycles.This is done on an automated cycler, which
can heat and cool the tubes with the reaction mixture in a very short time.
Target DNA
95oC
5’ 3’
3’ 5’
5’ 3’
3’ 5’
ANNEALING at 55°C.Ionic bonds → constantly formed and broken
between the single stranded primer & single stranded template.
More stable bonds last little bit longer and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template.
Once there are few bases built in, the ionic bond between template and the primer does not break any more.
~55oC
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’5’
Target DNA
A
B
primers
AB
EXTENSION AT 72°C: -This is the ideal temp., for the polymerase.Here, the primers that are on positions with
no exact match get loose again (because of the higher temp.,) and don’t give an extension of the fragment.
The bases complementary to the template are coupled to the primer on the 3’ side (the polymerase adds dNTPs from 5’ to 3’, reading the template from 3’ to 5’ side, bases are added complementary to the template).
PCR AND ITS REQUIREMENTS: -BASIC COMPONENTS AND REAGENTSDNA template.Two primers.Taq polymerase.Deoxynucleoside triphosphates (dNTPs).Buffer solution.Divalent cations.Monovalent cation.
PRIMERSShort artificial DNA strands- 18 -25 base pair
long
Starting point for DNA replication.
In natural DNA replication → primer → short RNA strand → primase → later removed or replaced with DNA by DNA polymerase.
TAQ POLYMERASEThermostable DNA polymerase →
thermophilic bacterium → Thermus aquaticus.
“Taq pol” or “Taq”.
Now, it has replaced E.coli DNA polymerase
PROCEDURE: -Initialization step.
Denaturation step.
Annealing step.
Extension/elongation step.
Final elongation.
PCR OPTIMIZATIONVarious techniques for PCR optimization have
been developed to improve PCR performance and to minimize failure.
Contamination and PCR. Hairpins. Polymerase errors. Nonspecific priming.